The present invention relates to a method of determining the enzymatic activity of the blood coagulation factor XIII using purified fibrin monomer as a substrate. Particularly, the present invention relates to a method of determining the enzymatic activity of the blood coagulation factor XIII by detecting the degree of fibrin cross-linking formed by the blood coagulation factor XIII, using fibrin monomer as a substrate, which are free of blood coagulation factor XIII.
The blood coagulation factor XIII, circulating in the blood as a zymogen, is activated by thrombin in the presence of calcium ion and it is one of the transglutaminase which catalyzes an acyl transfer reaction. The blood coagulation factor XIII catalyzes the covalent cross-linking of the fibrin molecules by forming the isopeptide bridge between lysine and glutamine residues of fibrin molecules. The substrates having specificity to the blood coagulation factor XIII are usually intra-platelet substances, monocyte, macromolecules on the surface of epithelial cells or macromolecules circulating in blood: fibrinxe2x80x94fibrin; fibrinxe2x80x94fibrin(fibrinogen); fibrinxe2x80x94xcex12-plasminogen inhibitory factor; fibrinxe2x80x94fibronectin; fibrinogenxe2x80x94collagen; fibrinxe2x80x94von Willebrand factor; thrombospondinxe2x80x94thrombospondin; blood coagulation factor Vxe2x80x94actin; von Willebrand factorxe2x80x94collagen; fibronectinxe2x80x94myosin; actinxe2x80x94myosin; gelsollinxe2x80x94fibrin or gelsollin; vincullinxe2x80x94fibrin (Karges, H. E., Clemens, R., Behring Inst. Mitt. 82: 43-58, 1988).
The blood coagulation factor XIII plays a role in hemostasis and wound healing process by forming fibrin clot which has strong mechanical strength and has an effect on cell proliferation, growth and metastasis of tumor cell and arteriosclerosis. It is known that the concentration of the blood coagulation factor XIII changes variously according to different pathologic states (inherited and acquired bleeding disease, chronic renal failure, malignant tumor, liver disease).
Since the blood coagulation factor XIII plays an important role in physiological field and the concentration of the blood coagulation factor XIII changes variously in different pathologic states, pathologic diagnosis by determining the enzymatic activity of the blood coagulation factor XIII is increasingly required in the clinical field. In addition, the assay method determining the enzymatic activity of the blood coagulation factor XIII is required in the inspection of pharmaceuticals containing the blood coagulation factor XIII as an effective agent. The assay method is also required for studying the characteristics of blood coagulation factor XIII.
Many previous techniques for the quantification method of the blood coagulation factor XIII have been developed which are, for example, clot lysis, SDS-PAGE of cross-linked fibrin, thromboelastography, binding of labeled or unlabeled amine, formation of ammonia in transamidation, detection of synthesized material and immunoassay (Karges, H. E., Clemens, R., Behring Inst. Mitt. 82: 43-58, 1988). But a rapid and simple assay method suitable for ordinary analysis has not been developed.
The conventional clot lysis method determines the enzymatic activity of the blood coagulation factor XIII in sample. The basis of this method is that, when dissolving agent (1% monocloroacetic acid) is added to fibrin clot, the enzymatic activity of the blood coagulation factor XIII in standard plasma diluted by minimal concentration (endpoint) enabling fibrin clot insoluble against dissolving agent, is same as the enzymatic activity of the blood coagulation factor XIII in sample diluted by maximal dilution factor making insoluble fibrin clot (Fibrin clot is formed by adding thrombin/calcium solution to the standard plasma (including blood coagulation factor XIII) and the sample, respectively diluted in appropriate dilution factor.). Therefore, the concentration (mU/ml) of the blood coagulation factor XIII in sample is calculated by multiplying the concentration of the blood coagulation factor XIII in standard plasma by dilution factor of sample making insoluble fibrin clot.
This assay method is simple, does not require expensive equipments and is easily used for determining the enzymatic activity of the blood coagulation factor XIII in clinical laboratories. However, there are problems in this method that the tight binding of the blood coagulation factor XIII to fibrinogen makes it difficult to remove the blood coagulation factor XIII from fibrinogen and that the blood coagulation factor XIII-free fibrinogen, the substrate of clot lysis method, is considerably expensive.
Accordingly, the present inventors have intensively studied to achieve a new simple method with reproducibility which can determine the enzymatic activity of the blood coagulation factor XIII rapidly and economically. This invention was completed by confirming that the enzymatic activity of the blood coagulation factor XIII can be determined economically and rapidly by detecting the degree of fibrin cross-linking formed by the blood coagulation factor XIII, using blood coagulation factor XIII-free fibrin monomer as a substrate.
The present invention provides a method of determining the enzymatic activity of the blood coagulation factor XIII by detecting the degree of fibrin crosslinking formed by the blood coagulation factor XIII using blood coagulation factor XIII-free fibrin monomer, as a substrate.
The present invention also provides a preparation method of fibrin monomer free of blood coagulation factor XIII by washing non-covalent fibrin polymer which is obtained from thrombin treated fibrinogen, with a suitable solution.